Terpenes are found in most organisms (microorganisms, animals and plants). These compounds are made up of five carbon units called isoprene units and are classified by the number of these units present in their structure. Thus monoterpenes, sesquiterpenes and diterpenes are terpenes containing 10, 15 and 20 carbon atoms respectively. Sesquiterpenes, for example, are widely found in the plant kingdom. Many terpene (e.g. sesquiterpene) molecules are known for their flavor and fragrance properties and their cosmetic, medicinal and antimicrobial effects. Numerous terpene (e.g. sesquiterpene) hydrocarbons and terpenoids (e.g. sesquiterpenoids) have been identified.
Biosynthetic production of terpenes involves enzymes called terpene synthases. These enzymes convert an acyclic terpene precursor in one or more terpene products. In particular, diterpene synthases produce diterpenes by cyclization of the precursor geranylgeranyl pyrophosphate (GGPP). The cyclization of GGPP often requires two enzyme polypeptides, a type I and a type II diterpene synthase working in combination in two successive enzymatic reactions. The type II diterpene synthases catalyze a cyclization/rearrangement of GGPP initiated by the protonation of the terminal double bond of GGPP leading to a cyclic diterpene diphosphate intermediate. This intermediate is then further converted by a type I diterpene synthase catalyzing an ionization initiated cyclization.
Diterpene synthases are present in the plants and other organisms and use substrates such as geranlygeranyl diphosphate but they have different product profiles. Genes and cDNAs encoding diterpene synthases have been cloned and the corresponding recombinant enzymes characterized.
Copalyl diphosphate synthases and sclareol synthases are enzymes that occur in plants. Hence, it is desirable to discover and use these enzymes and variants in biochemical processes to generate (+)-manool